Anti-hepatitis b virus ribozymal nucleic acid

Abstract

This invention relates to ribozymes which cleave Hepatitis B Virus (HBV) at CUC sites. Suitable ribozymes may, for example, cleave at GGCUCUCUCGUCCC, CCUCAGCUCUGUAUCG or GAGGACUCUUGGA recognition sequences in HBV RNA. Ribozymal DNA, vector systems and pharmaceutical compositions are provided which may be useful, for example, in the treatment of HBV infection.

1 . Ribozymal DNA transcribable to an RNA having a catalytic sequence in two parts, including an intervening, stabilizing stem having the sequence GCGCGAAAGCGC (SEQ ID NO:38) located between said two parts, wherein said RNA will cleave HBV RNA at a CUC site. 
     
     
         2 . (canceled) 
     
     
         3 . The ribozymal DNA according to  claim 1 , having a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 (MAZ1), SEQ ID NO:5 (SGIII) and SEQ ID NO:6 (DGII). 
     
     
         4 - 5 . (canceled) 
     
     
         6 . The ribozymal DNA according to  claim 1 , which is transcribable to RNA which binds to an HBV recognition site having a nucleotide sequence selected from the group consisting of GGCUCUCUCGUCCC (MAZ1) (SEQ ID NO:1), CCUCAGCUCUGUAUCG (SGIII) (SEQ ID NO:2) and GAGGACUCUUGGA (DGII) (SEQ ID NO:3). 
     
     
         7 - 9 . (canceled) 
     
     
         10 . The ribozymal DNA according to  claim 1 , which, when transcribed to RNA, will cleave at three sites in a target RNA sequence present in HBV RNA and which contains recognition sequences as follows (5′ to 3′): 
       
         
           
                 
                 
               
                   (SEQ ID NO:42) 
                     
                 
                 
                 
                 
               
                     
                   GGCTCTCTCGTCCC for ribozyme MAZI 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO:43) 
                     
                 
                 
                 
                 
               
                     
                   CCTCAGCTCTGTATCG for ribozyme SGIII 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO:41) 
                     
                 
                 
                 
                 
               
                     
                   GAGGACTCTTGGA for ribozyme DGII. 
                     
                 
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
               
            
           
         
       
     
     
         11 . A vector system comprising at least one DNA vector, the at least one DNA vector containing a target-cleaving ribozymal DNA sequence under control of a promoter effective in human cells and which, upon transcription to RNA will cleave the RNA transcribed from a HBV target genome at CUC sites therein. 
     
     
         12 . The vector system according to  claim 11 , containing at least one target-cleaving ribozymal sequence for one or more of the ribozymes MAZI, DGII and SGIII cleaving mRNA transcribed from HBV target genome. 
     
     
         13 . The vector system according to  claim 11 , comprising at least two DNA vectors, wherein a first vector contains a first promoter that allows gene expression in human cells, operably linked to a gene which is expressible to produce an activator protein capable of acting on a second promoter, wherein a second vector contains the second promoter operably linked to one or more of target-cleaving ribozymal DNA sequences for one or more RNA sequences selected from the group consisting of MAZI RNA, DGII RNA, and SGIII RNA to target HBV RNA. 
     
     
         14 . The vector system according to  claim 13 , comprising at least three DNA vectors, wherein the second vector contains target-cleaving ribozymal DNA for MAZI, and wherein a third vector contains target-cleaving ribozymal DNA for SGIII cleaving mRNA transcribed from the targeted HBV RNA. 
     
     
         15 . The vector system according to  claim 14 , comprising at least four DNA vectors, wherein the second vector contains target-cleaving ribozymal DNA for MAZI, the third vector contains target-cleaving ribozymal DNA for SGIII, and wherein a fourth vector contains target-cleaving ribozymal DNA for DGII cleaving mRNA transcribed from the targeted HBV RNA. 
     
     
         16 . (canceled) 
     
     
         17 . The vector system according to  claim 13 , wherein the second promoter is a T7 polymerase promoter and the activator protein is a T7 polymerase. 
     
     
         18 - 19 . (canceled) 
     
     
         20 . The vector system according to  claim 11 , wherein the ribozymal DNA sequence further comprises, downstream of the target-cleaving ribozymal sequence, a 3′-autocatalytic hammerhead ribozymal DNA sequence, so that when the ribozymal DNA is transcribed to RNA, it has a representable form as a double hammerhead, having first and second stems of a target-cleaving ribozyme which targets HBV RNA and first and second stems of 3′-autocatalytic ribozyme. 
     
     
         21 - 22 . (canceled) 
     
     
         23 . The vector system acid according to  claim 20 , wherein the target-cleaving ribozyme sequence comprises in order (5′ to 3′):
 a first structure-stabilizing stem loop;   a first target-recognition sequence;   a first catalytic sequence;   a second structure-stabilizing stem loop;   a second catalytic sequence; and   a second target-recognition sequence.   
     
     
         24 . The vector system according to  claim 11 , wherein the target-cleaving ribozymal DNA sequence, when transcribed to RNA, will cleave a target RNA sequence present in HBV RNA, and which contains recognition sequences (5′ to 3′): 
       
         
           
                 
                 
               
                   (SEQ ID NO:42) 
                     
                 
                 
                 
                 
               
                     
                   GGCTCTCTCGTCCC for ribozyme MAZI 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO:43) 
                     
                 
                 
                 
                 
               
                     
                   CCTCAGCTCTGTATCG for ribozyme SGIII 
                     
                 
                     
                     
                 
                 
                 
               
                   (SEQ ID NO:41) 
                     
                 
                 
                 
                 
               
                     
                   GAGGACTCTTGGA for ribozyme DGII. 
                     
                 
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
               
            
           
         
       
     
     
         25 . A lentivirus containing a vector system according to  claim 11 . 
     
     
         26 . A pharmaceutically acceptable carrier containing ribozymal DNA according to  claim 1 , a vector system defined in  claim 11 , or a lentivirus according to  claim 25 . 
     
     
         27 . The carrier according to  claim 26  in the form of liposomes. 
     
     
         28 . (canceled) 
     
     
         29 . A method of treating a disease associated with HBV infection, comprising administering the ribozymal DNA according to  claim 1 , a vector system according to  claim 11 , or a lentivirus according to  claim 25  to an individual in need thereof. 
     
     
         30 - 31 . (canceled) 
     
     
         32 . Ribozymal DNA comprising: (1) a target-cleaving hammerhead ribozymal DNA sequence under control of a promoter effective in human cells and which, upon transcription to RNA, will cleave mRNA transcribed from a target gene encoding an HBV genome, and downstream thereof; and (2) a 3′-autocatalytic hammerhead ribozymal DNA sequence, so that when the ribozymal DNA is transcribed to RNA, it has a form represented as a double hammerhead, having first and second stems of a target-cleaving ribozyme which targets HBV RNA and first and second stems of 3′-autocatalytic ribozyme, together with a common third system joining the two hammerheads. 
     
     
         33 . (canceled)